The present invention relates generally to methods for stimulating cells, and more particularly, to methods to concentrate and stimulate cells that maximizes stimulation of such cells. The present invention also relates to compositions of cells, including stimulated T-cells having specific phenotypic characteristics.
Many cells are activated or regulated via receptors embedded in lipid rafts found in cell surface membranes. See K. Simons and D. Toomre, Nature Rev. 1:31, 2000. Lipid rafts form concentrating platforms for individual receptors that are activated by ligand binding. Lipid rafts are involved in cellular signaling processes, including immunoglobulin E signaling during the allergic immune response, glial-cell-derived neurotrophic factor signaling important for the development and maintenance of the nervous system, Ras signaling, central to many signal transduction processes, and T-cell antigen receptor (TCR) signaling.
The T-cell antigen receptor (TCR) is a multisubunit immune recognition receptor that associates with the CD3 complex and binds to peptides presented by the major histocompatibility complex (MHC) class I and II proteins on the surface of antigen-presenting cells (APCs). Binding of TCR to the antigenic peptide on the APC is the central event in T-cell activation, which occurs at an immunological synapse at the point of contact between the T-cell and the APC. Moreover, data suggest that clustering of lipid rafts is essential to the formation of the immunological synapse. Krawczyk et al., Immunity 13(4):463-73, 2000.
To sustain T-cell activation, T lymphocytes typically require a second co-stimulatory signal. Co-stimulation is typically necessary for a T helper cell to produce sufficient cytokine levels that induce clonal expansion. Bretscher, Immunol. Today 13:74, 1992; June et al., Immunol. Today 15:321, 1994. The major co-stimulatory signal occurs when a member of the B7 family ligands (CD80 (B7.1) or CD86 (B7.2)) on an activated antigen-presenting cell (APC) binds to CD28 on a T-cell.
Methods of stimulating the expansion of certain subsets of T-cells have the potential to generate a variety of T-cell compositions useful in immunotherapy. Successful immunotherapy can be aided by increasing the reactivity and quantity of T-cells by efficient stimulation.
The various techniques available for expanding human T-cells have relied primarily on the use of accessory cells and/or exogenous growth factors, such as interleukin-2 (IL-2). IL-2 has been used together with an anti-CD3 antibody to stimulate T-cell proliferation, predominantly expanding the CD8+ subpopulation of T-cells. Both APC signals are thought to be required for optimal T-cell activation, expansion, and long-term survival of the T-cells upon re-infusion. The requirement for MHC-matched APCs as accessory cells presents a significant problem for long-term culture systems because APCs are relatively short-lived. Therefore, in a long-term culture system, APCs must be continually obtained from a source and replenished. The necessity for a renewable supply of accessory cells is problematic for treatment of immunodeficiencies in which accessory cells are affected. In addition, when treating viral infection, if accessory cells carry the virus, the cells may contaminate the entire T-cell population during long-term culture.
In the absence of exogenous growth factors or accessory cells, a co-stimulatory signal may be delivered to a T-cell population, for example, by exposing the cells to a CD3 ligand and a CD28 ligand attached to a solid phase surface, such as a bead. See C. June, et al. (U.S. Pat. No. 5,858,358); C. June et al. WO 99/953823. While these methods are capable of achieving therapeutically useful T-cell populations, increased robustness and ease of T-cell preparation remain less than ideal.
In addition, the methods currently available in the art have not focused on short-term expansion of T-cells or obtaining a more robust population of T-cells and the beneficial results thereof and/or the expansion of particular T-cell subclasses/phenotypes. Furthermore, the applicability of expanded T-cells has been limited to only a few disease states. For maximum in vivo effectiveness, theoretically, an ex vivo- or in vivo-generated, activated T-cell population should be in a state that can maximally orchestrate an immune response to cancer, infectious disease, or other disease states. The present invention provides methods to generate an increased number of more highly activated and more pure T-cells that have surface receptor and cytokine production characteristics that appear more healthy and natural than other expansion methods.
In addition, the present invention provides compositions of phenotypically tailored cell populations of any target cell, including T-cell populations and parameters for producing the same, as well as providing other related advantages.
The present invention generally provides methods for stimulating cells, and more particularly, provides a novel method to concentrate and stimulate cells that maximizes stimulation of such cells. In one aspect the present invention provides methods for stimulating a population of T-cells by simultaneous T-cell concentration and cell surface moiety ligation that comprises providing a population of cells wherein at least a portion thereof comprises T-cells, contacting the population of cells with a surface, wherein the surface has attached thereto one or more agents that ligate a cell surface moiety of at least a portion of the T-cells and stimulates at least that portion of T-cells or a subpopulation thereof and applying a force that predominantly drives T-cell concentration and T-cell surface moiety ligation, thereby inducing T-cell stimulation.
In one embodiment of the methods the surface has attached thereto a first agent that ligates a first cell surface moiety of a T-cell; and the same or a second surface has attached thereto a second agent that ligates a second moiety of said T-cell, wherein said ligation by the first and second agent induces proliferation of said T-cell. In related embodiments the surface may be biocompatible, natural or synthetic, comprise a polymer, comprise collagen, purified proteins, purified peptides, polysaccharides, glycosaminoglycans, or extracellular matrix compositions. In certain embodiments, the polysaccharides are selected from chitosan, alginate, dextran, hyaluronic acid, and cellulose and the polymer is selected from polystyrene, polyesters, polyethers, polyanhydrides, polyalkylcyanoacrylates, polyacrylamides, polyorthoesters, polyphosphazenes, polyvinylacetates, block copolymers, polypropylene, polytetrafluoroethylene (PTFE), or polyurethanes. In yet other embodiments, the polymer may comprise lactic acid or a copolymer. While in still yet other embodiments, the polymer may be a copolymer. Such copolymers can be a variety of known copolymers and may include lactic acid and/or glycolic acid (PLGA).
With respect to biocompatible surfaces, such surfaces may be biodegradable or non-biodegradable. In related embodiments, while not limited thereto, the non-biodegradable surfaces may comprise poly(dimethysiloxane) and/or poly(ethylene-vinyl acetate). Further, the biocompatible surface, while not limited thereto, may include collagen, metal, hydroxyapatite, glass, aluminate, bioceramic materials, hyaluronic acid polymers, alginate, acrylic ester polymer, lactic acid polymer, glycolic acid polymer, lactic acid/glycolic acid polymer, purified proteins, purified peptides, and/or extracellular matrix compositions.
In still yet further embodiments, the biocompatible surface is associated with an implantable device. The implantable device may be any that is desired to be used and may include a stent, a catheter, a fiber, a hollow fiber, a patch, or a suture. In related embodiments the surface may be glass, silica, silicon, collagen, hydroxyapatite, hydrogels, PTFE, polypropylene, polystyrene, nylon, or polyacrylamide. Yet additional embodiments include wherein the surface comprises a lipid, a plate, a bag, a rod, a pellet, a fiber, or a mesh. Other embodiments include wherein the surface is a particle and additionally wherein the particle comprises a bead, a microsphere, a nanoparticle, or a colloidal particle. Particle and bead sizes may also be chosen and may have a variety of sizes including wherein the bead is about 5 nanometers to about 500 microns in diameter.
In other embodiments, the agents used in the methods can be independently selected from a protein ligand, a natural ligand, or a synthetic ligand. Further, the agents may also comprise an antibody, an antibody fragment, a peptide, a polypeptide, a glycopeptide, a soluble receptor, a steroid, a hormone, a mitogen, an antigen, a superantigen, a growth factor, a cytokine, a lectin, a viral protein, an adhesion molecule, or a chemokine. In specific embodiments, at least one agent is an antibody or an antibody fragment. While in yet other embodiments, a first agent is an antibody and a fragment thereof, and a second agent is an antibody or a fragment thereof. It would of course be understood that the first and second agents could either be the same or different antibodies.
In selected embodiments the first agent is an anti-CD3 antibody, an anti-CD2 antibody, or an antibody fragment of an anti-CD3 or anti-CD2 antibody. Further selected embodiments include wherein the second agent is an anti-CD28 antibody or antibody fragment thereof. Further embodiments include wherein the second agent comprises a natural ligand for CD28, such as, e.g., B7-1 or B7-2. In addition, other stimulatory agents could be used.
In certain embodiments, the force used to drive the cells may include a variety of forces that function similarly, and include a force greater than gravitational force, a hydraulic force, a filtration force generated by transmembrane pressure, a centrifugal force, or a magnetic force. When magnetic forces are used, some embodiments utilize a magnetic force that is generated by a magnet having a magnetic field strength ranging from between about 200 gauss to about 12,000 gauss at the surface of the magnet.
Another embodiment includes surfaces wherein the surface is a surface of a paramagnetic particle. While in embodiments utilizing surfaces including a surface of a paramagnetic particle the agents attachment to the surface may be covalent, noncovalent, electrostatic, inter-molecular adhesion, or hydrophobic.
In still yet other embodiments the T-cells that are ligated are separated from the T-cells that are not ligated. While in other embodiments the T-cells ameliorate immune response dysfunction.
Other aspects that may be combined with the embodiments above include, for example methods for stimulation of T-cells by simultaneous cell surface moiety ligation and T-cell aggregation comprising providing a cell population comprising T-cells, contacting said cell population with a surface, wherein said surface has attached thereto one or more ligands specific for a cell surface moiety, applying a force that drives concentration of T-cells and surface and incubating said cells for a period of time sufficient to achieve desired stimulation. In related embodiments the time sufficient to achieve desired stimulation may range from 1 minute to 10 days and all integer values, in between. In certain embodiments, the time range may be from about 1 day to about 8 days, while in yet other embodiments the time range may be from about 3 days to about 5 days, or from about 1 day to about 5 days. In related embodiments the incubation temperature may range from about 2 to about 38xc2x0 C.
Further embodiments that can be used with all the recited methods include wherein the surface is selected from glass, silica, silicon, collagen, hydroxyapatite, hydrogels, PTFE, polypropylene, polystyrene, nylon, dextran, or polyacrylamide or mixtures of any of these. Further, embodiments include prior to or concurrently with any steps noted above, separating T-cells concentrated with surface from non-concentrated cells.
In other aspects methods of inducing T-cell activation in vivo are provided, comprising providing paramagnetic particles to an animal, said particles having attached thereto, ligands specific for a T-cell surface moiety that induces T-cell activation; applying a magnetic field to a discrete region of the animal; and thereby inducing localization and activation of T-cells bound to said particles at said discrete region.
An additional aspect is provided that includes methods for stimulating a population of target cells by simultaneous target cell concentration and target cell surface moiety ligation, comprising providing a population of cells wherein at least a portion thereof comprises target cells contacting said population of cells with a surface, wherein said surface has attached thereto one or more agents that ligate a cell surface moiety of at least a portion of said target cells and stimulates at least said portion of target cells, applying a force that predominantly drives target cell concentration and target cell surface moiety ligation, thereby inducing target cell stimulation.
In certain embodiments, the methods described herein utilize a surface that has attached thereto a first agent that ligates a first cell surface moiety of a target cell; and the same or a second surface has attached thereto a second agent that ligates a second moiety of said target cell, wherein said ligation by the first and second agent induces signal transduction in said target cell.
As noted previously, the surface may include a variety of components including collagen, purified proteins, purified peptides, polysaccharides, glycosaminoglycans, and/or extracellular matrix compositions. Some polysaccharides that are utilized in specific embodiments may include chitosan, alginate, dextran, hyaluronic acid, and/or cellulose. Further, polymers as noted above and applicable to all methods may be selected from polyesters, polyethers, polyanhydrides, polyalkylcyanoacrylates, polyacrylamides, polyorthoesters, polyphosphazenes, polyvinylacetates, block copolymers, polypropylene, polytetrafluoroethylene (PTFE), and/or polyurethanes and mixtures thereof.
In other aspects the methods are provided for stimulation of target cells by cell surface moiety ligation and target cell concentration, comprising providing a cell population comprising target cells, contacting said cell population with a surface, wherein said surface has attached thereto one or more ligands specific for a cell surface moiety, applying a force that drives concentration of target cells and concentration of said cells on said surface and incubating said cells for a period of time sufficient to achieve desired stimulation.
In related embodiments the target cells may be T-cells, B-cells, or stem cells.
Other aspects provide methods of inducing target cell stimulation in vivo, comprising providing paramagnetic particles to an animal, said particles having attached thereto, ligands specific for a target cell surface moiety that induces target cell stimulation; applying a magnetic field to a discrete region of the animal; and thereby inducing localization and stimulation of the target cells bound to said particles at said discrete region.
Still other aspects are provided which include methods for inducing receptor polarization in receptor bearing cells comprising providing a cell population, contacting said cell population with a solid surface, wherein said solid surface has attached thereto one or more ligands specific for a cell surface receptor present on at least a portion of said cell population and applying a force that drives cell concentration and cell surface receptor ligation.
Other aspects include methods for inducing aggregation of cell surface molecules, comprising providing a population of cells having a target cell surface molecule, contacting said population of cells with a solid surface, wherein said solid surface has attached thereto a ligand for at least one target cell surface molecule, applying a force that drives aggregation of targeted cell surface molecules.
In certain embodiments the cell population comprises lymphocytes.
In yet other certain embodiments the receptor or cell surface moiety binding leads to down regulation or suppression of a cellular event. Related embodiments include wherein the receptor binding leads to up regulation or activation of a cellular event, which may include, for example, receptor mediated signal transduction.
Another embodiment of the invention envisions the use of a force to drive concentration or orientation of cell surface moieties.
Yet additional embodiments of the present invention provide phenotypically tailored target cell populations and/or compositions including T-cell compositions. In addition, methods are provided for activating such cells by ligating a cell surface moiety. Further provided are methods for inducing a population of T-cells to proliferate, comprising contacting the T-cells with a solid surface for a period of time of between about two hours and about nine days, the solid surface having immobilized thereon a first agent and second agent, and wherein the first agent provides an activation signal and the second agent provides a co-stimulatory signal to said T-cells.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings.